Sporulation is a multi-stage developmental process that is responsible for the conversion of a growing cell into a dormant cell type, known as a spore or endospore. Spores are adapted for dispersal and survival for an extended period of time in unfavourable conditions and form part of the life cycle of many plants, algae and bacteria, such as the Bacillus species.
The primary regulator for entry into sporulation is the DNA-binding protein Spo0A (stage 0 sporulation protein A), which is a member of the response regulator family of transcription factors. Numerous other genes, including genes which encode five histidine autokinases (KinA, KinB, KinC, KinD and KinE) and two response proteins (Spo0B and Spo0F), are also involved in the control of the initiation of sporulation (Molle et al.; Mol. Microbial.; 2003, 50(5):1683-1701). The activity of Spo0A is governed by a multi-component phosphorelay, which recognises and integrates environmental signals to initiate sporulation (Trach K A, et al; Mol. Microbiol. 1993; 8(1):69-79). Upon phosphorylation of its regulatory N-terminal domain, Spo0A-P binds to a DNA sequence element known as the “0A-box” which activates genes involved in sporulation. Deletion of the C-terminal domain of Spo0A, which is inactive until the N-terminus has been phosphorylated, has been shown to result in a sporulation-negative phenotype (Rowe-Magnus DA, et al; J. Bacteriol.; 2000; 182(15):4352-4355).
Spo0A has also been found to influence, directly or indirectly, the activation or repression of expression of over 500 genes in B. subtilis, and therefore indirectly mediates the global pattern of gene transcription via regulatory genes under its control (Molle et al.; Mol. Microbiol.; 2003, 50(5):1683-1701).
Sporulation is subject to catabolite repression, whereby the presence of glucose or other readily metabolized carbon sources inhibits sporulation by wild-type cells. In particular, glucose is known to repress the transcription of spo0A and spo0F (Myseliwiec, TH et al; J. Bacterial.; 1991; 173(6):1911-1919). In a commercial fermentation process spores are undesirable for two main reasons:    1. Sporulation pauses active metabolism by an organism resulting in a reduction or cessation of the formation of a desired metabolic product; and    2. Sporulating microorganisms are more difficult to handle and control containment, therefore it is desirable to avoid the survival of commercial process microorganisms for environmental reasons, including health and safety, and also to prevent the uncontrolled release of the commercial strain.
The general process by which bacteria metabolise suitable substrates is glycolysis, which is a sequence of reactions that converts glucose into pyruvate with the generation of ATP. The fate of pyruvate in the generation of metabolic energy varies depending on the microorganism and the environmental conditions. The four principal reactions of pyruvate are illustrated in FIG. 5.
First, under aerobic conditions, many microorganisms will generate energy using the citric acid cycle and the conversion of pyruvate into acetyl coenzyme A, catalysed by pyruvate dehydrogenase (PDH).
Second, under anaerobic conditions, certain ethanologenic organisms can carry out alcoholic fermentation by the decarboxylation of pyruvate into acetaldehyde, catalysed by pyruvate decarboxylase (PDC) and the subsequent reduction of acetaldehyde into ethanol by NADH, catalysed by alcohol dehydrogenase (ADH).
A third reaction, which also occurs in anaerobic conditions, is the conversion of pyruvate to acetyl CoA, catalysed by pyruvate formate lyase (PFL). Acetyl CoA is subsequently converted into acetaldehyde by the enzyme acetaldehyde dehydrogenase (AcDH) and ethanol is produced by the reduction of acetaldehyde catalysed by ADH.
A fourth process is the conversion of pyruvate into lactate which occurs through catalysis by lactate dehydrogenase (LDH).
There has been much interest in using microorganisms for the production of ethanol using either microorganisms that undergo anaerobic fermentation naturally or through the use of recombinant microorganisms which incorporate the pyruvate decarboxylase and alcohol dehydrogenase genes.
WO2008/038019 discloses microorganisms which comprise modifications to inactivate the native LDH and PFL genes and up-regulate the PDC, PDH and ADH genes in order to promote the formation of ethanol.
There is a need for further improvements to the production of ethanol from microorganisms.